Activation of human RNA lariat debranching enzyme Dbr1 by binding protein TTDN1 occurs though an intrinsically disordered C-terminal domain.

In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.

Crystal Structure of the RNA Lariat Debranching Enzyme Dbr1 with Hydrolyzed Phosphorothioate RNA Product.

The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2'-5' phosphodiester bond in RNA lariats produced by the spliceosome. Here, we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) that contain a 2'-5'-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to cocrystallize a phosphorothioate-branched RNA (PS-bRNA) with wild-type Entamoeba histolytica Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond. The crystal structure revealed EhDbr1 in a product-bound state, with the hydrolyzed 2'-5' fragment of the PS-bRNA mimicking the binding mode of the native bRNA substrate. These findings suggest that product inhibition may contribute to the kinetic mechanism of Dbr1. We show that Dbr1 enzymes cleave phosphorothioate linkages at rates ∼10,000-fold more slowly than native phosphate linkages. This new product-bound crystal structure offers atomic details, which can aid inhibitor design. Dbr1 inhibitors could be therapeutic or investigative compounds for human diseases such as human immunodeficiency virus (HIV), amyotrophic lateral sclerosis (ALS), cancer, and viral encephalitis.

A pH Switch Controls Zinc Binding in Tomato Copper-Zinc Superoxide Dismutase.

Copper-zinc superoxide dismutase (SOD1) is a major antioxidant metalloenzyme that protects cells from oxidative damage by superoxide anions (O2-). Structural, biophysical, and other characteristics have in the past been compiled for mammalian SOD1s and for the highly homologous fungal and bovine SOD1s. Here, we characterize the biophysical properties of a plant SOD1 from tomato chloroplasts and present several of its crystal structures. The most unusual of these structures is a structure at low pH in which tSOD1 harbors zinc in the copper-binding site but contains no metal in the zinc-binding site. The side chain of D83, normally a zinc ligand, adopts an alternate rotameric conformation to form an unusual bidentate hydrogen bond with the side chain of D124, precluding metal binding in the zinc-binding site. This alternate conformation of D83 appears to be responsible for the previously observed pH-dependent loss of zinc from the zinc-binding site of SOD1. Titrations of cobalt into apo tSOD1 at a similar pH support the lack of an intact zinc-binding site. Further characterization of tSOD1 reveals that it is a weaker dimer relative to human SOD1 and that it can be activated in vivo through a copper chaperone for the SOD1-independent mechanism.

Why does oxamniquine kill Schistosoma mansoni and not S. haematobium and S. japonicum?

Human schistosomiasis is a disease which globally affects over 229 million people. Three major species affecting humans are Schistosoma mansoni, S. haematobium and S. japonicum. Previous treatment of S. mansoni includes the use of oxamniquine (OXA), a prodrug that is enzymatically activated in S. mansoni but is ineffective against S. haematobium and S. japonicum. The OXA activating enzyme was identified and crystallized, as being a S. mansoni sulfotransferase (SmSULT). S. haematobium and S. japonicum possess homologs of SmSULT (ShSULT and SjSULT) begging the question; why does oxamniquine fail to kill S. haematobium and S. japonicum adult worms? Investigation of the molecular structures of the sulfotransferases indicates that structural differences, specifically in OXA contact residues, do not abrogate OXA binding in the active sites as previously hypothesized. Data presented argue that the ability of SULTs to sulfate and thus activate OXA and its derivatives is linked to the ability of OXA to fit in the binding pocket to allow the transfer of a sulfur group.

Oxamniquine resistance alleles are widespread in Old World Schistosoma mansoni and predate drug deployment.

Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni.

Experimental Mutations in Superoxide Dismutase 1 Provide Insight into Potential Mechanisms Involved in Aberrant Aggregation in Familial Amyotrophic Lateral Sclerosis.

Mutations in more than 80 different positions in superoxide dismutase 1 (SOD1) have been associated with amyotrophic lateral sclerosis (fALS). There is substantial evidence that a common consequence of these mutations is to induce the protein to misfold and aggregate. How these mutations perturb native structure to heighten the propensity to misfold and aggregate is unclear. In the present study, we have mutagenized Glu residues at positions 40 and 133 that are involved in stabilizing the ß-barrel structure of the native protein and a critical Zn binding domain, respectively, to examine how specific mutations may cause SOD1 misfolding and aggregation. Mutations associated with ALS as well as experimental mutations were introduced into these positions. We used an assay in which mutant SOD1 was fused to yellow fluorescent protein (SOD1:YFP) to visualize the formation of cytosolic inclusions by mutant SOD1. We then used existing structural data on SOD1, to predict how different mutations might alter local 3D conformation. Our findings reveal an association between mutant SOD1 aggregation and amino acid substitutions that are predicted to introduce steric strain, sometimes subtly, in the 3D conformation of the peptide backbone.

Design, Synthesis, and Characterization of Novel Small Molecules as Broad Range Antischistosomal Agents.

Schistosomiasis is a major human parasitic disease afflicting more than 250 million people, historically treated with chemotherapies praziquantel or oxamniquine. Since oxamniquine is species-specific, killing Schistosoma mansoni but not other schistosome species (S. haematobium or S. japonicum) and evidence for drug resistant strains is growing, research efforts have focused on identifying novel approaches. Guided by data from X-ray crystallographic studies and Schistosoma worm killing assays on oxamniquine, our structure-based drug design approach produced a robust structure-activity relationship (SAR) program that identified several new lead compounds with effective worm killing. These studies culminated in the discovery of compound 12a, which demonstrated broad-species activity in killing S. mansoni (75%), S. haematobium (40%), and S. japonicum (83%).

Localized Induction of Wild-Type and Mutant Alpha-Synuclein Aggregation Reveals Propagation along Neuroanatomical Tracts.

Misfolded alpha-synuclein (αS) may exhibit a number of characteristics similar to those of the prion protein, including the apparent ability to spread along neuroanatomical connections. The demonstration for this mechanism of spread is largely based on the intracerebral injections of preaggregated αS seeds in mice, in which it cannot be excluded that diffuse, surgical perturbations and hematogenous spread also contribute to the propagation of pathology. For this reason, we have utilized the sciatic nerve as a route of injection to force the inoculum into the lumbar spinal cord and induce a localized site for the onset of αS inclusion pathology. Our results demonstrate that mouse αS fibrils (fibs) injected unilaterally in the sciatic nerve are efficient in inducing pathology and the onset of paralytic symptoms in both the M83 and M20 lines of αS transgenic mice. In addition, a spatiotemporal study of these injections revealed a predictable spread of pathology to brain regions whose axons synapse directly on ventral motor neurons in the spinal cord, strongly supporting axonal transport as a mechanism of spread of the αS inducing, or seeding, factor. We also revealed a relatively decreased efficiency for human αS fibs containing the E46K mutation to induce disease via this injection paradigm, supportive of recent studies demonstrating a diminished ability of this mutant αS to undergo aggregate induction. These results further demonstrate prion-like properties for αS by the ability for a progression and spread of αS inclusion pathology along neuroanatomical connections.IMPORTANCE The accumulation of alpha-synuclein (αS) inclusions is a hallmark feature of Parkinson's disease (PD) and PD-related diseases. Recently, a number of studies have demonstrated similarities between the prion protein and αS, including its ability to spread along neuroanatomical tracts throughout the central nervous system (CNS). However, there are caveats in each of these studies in which the injection routes used had the potential to result in a widespread dissemination of the αS-containing inocula, making it difficult to precisely define the mechanisms of spread. In this study, we assessed the spread of pathology following a localized induction of αS inclusions in the lumbar spinal cord following a unilateral injection in the sciatic nerve. Using this paradigm, we demonstrated the ability for αS inclusion spread and/or induction along neuroanatomical tracts within the CNS of two αS-overexpressing mouse models.

Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

Mycoplasma pneumoniae Community-Acquired Respiratory Distress Syndrome Toxin Uses a Novel KELED Sequence for Retrograde Transport and Subsequent Cytotoxicity.

Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, M. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity.IMPORTANCEMycoplasma pneumoniae, a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designated community-acquired respiratory distress syndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with M. pneumoniae infection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminate M. pneumoniae-associated airway and extrapulmonary pathologies.

Structure-Based Design and Synthesis of Potent and Selective Matrix Metalloproteinase 13 Inhibitors.

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (10d and (S)-17b) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound 5 in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., 10a and 10b). After demonstrating that the pharmacophores of the chelating inhibitors (S)-10a, (R)-10a, and 10b were binding within the MMP-13 active site, the Zn2+ chelating unit was replaced with nonchelating polar residues that bridged over the Zn2+ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors 10d and (S)-17b, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn2+ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca2+-dependent manner. In these cases, membrane association is triggered by Ca2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd2+, in lieu of Ca2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd2+-complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca2+ ion binding to the C2 domain loop regions.

Structural and enzymatic insights into species-specific resistance to schistosome parasite drug therapy.

The antischistosomal prodrug oxamniquine is activated by a sulfotransferase (SULT) in the parasitic flatworm Schistosoma mansoni. Of the three main human schistosome species, only S. mansoni is sensitive to oxamniquine therapy despite the presence of SULT orthologs in Schistosoma hematobium and Schistosoma japonicum The reason for this species-specific drug action has remained a mystery for decades. Here we present the crystal structures of S. hematobium and S. japonicum SULTs, including S. hematobium SULT in complex with oxamniquine. We also examined the activity of the three enzymes in vitro; surprisingly, all three are active toward oxamniquine, yet we observed differences in catalytic efficiency that implicate kinetics as the determinant for species-specific toxicity. These results provide guidance for designing oxamniquine derivatives to treat infection caused by all species of schistosome to combat emerging resistance to current therapy.

Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site.

Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.

An engineered transforming growth factor ß (TGF-ß) monomer that functions as a dominant negative to block TGF-ß signaling.

The transforming growth factor ß isoforms, TGF-ß1, -ß2, and -ß3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-ß pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-ßs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-ß monomer, lacking the heel helix, a structural motif essential for binding the TGF-ß type I receptor (TßRI) but dispensable for binding the other receptor required for TGF-ß signaling, the TGF-ß type II receptor (TßRII), as an alternative therapeutic modality for blocking TGF-ß signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-ß monomers and bound TßRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-ß signaling with a Ki of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TßRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TßRI, enabled it to bind endogenous TßRII but prevented it from binding and recruiting TßRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-ß signaling and may inform similar modifications of other TGF-ß family members.

Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition.

We have developed fluorescent 2',5' branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5' arm of the bRNA that is quenched by a dabcyl moiety on the 2' arm. Dbr1-mediated hydrolysis of the 2',5' linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2',5'-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1.

Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2',5'- and 3',5'-phosphodiester linkages. The 2',5'-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s-1 Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s-1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the ß-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.

Distinct conformers of transmissible misfolded SOD1 distinguish human SOD1-FALS from other forms of familial and sporadic ALS.

Evidence of misfolded wild-type superoxide dismutase 1 (SOD1) has been detected in spinal cords of sporadic ALS (sALS) patients, suggesting an etiological relationship to SOD1-associated familial ALS (fALS). Given that there are currently a number of promising therapies under development that target SOD1, it is of critical importance to better understand the role of misfolded SOD1 in sALS. We previously demonstrated the permissiveness of the G85R-SOD1:YFP mouse model for MND induction following injection with tissue homogenates from paralyzed transgenic mice expressing SOD1 mutations. This prompted us to examine whether WT SOD1 can self-propagate misfolding of the G85R-SOD1:YFP protein akin to what has been observed with mutant SOD1. Using the G85R-SOD1:YFP mice, we demonstrate that misfolded conformers of recombinant WT SOD1, produced in vitro, induce MND with a distinct inclusion pathology. Furthermore, the distinct pathology remains upon successive passages in the G85R-SOD1:YFP mice, strongly supporting the notion for conformation-dependent templated propagation and SOD1 strains. To determine the presence of a similar misfolded WT SOD1 conformer in sALS tissue, we screened homogenates from patients diagnosed with sALS, fALS, and non-ALS disease in an organotypic spinal cord slice culture assay. Slice cultures from G85R-SOD1:YFP mice exposed to spinal homogenates from patients diagnosed with ALS caused by the A4V mutation in SOD1 developed robust inclusion pathology, whereas spinal homogenates from more than 30 sALS cases and various controls failed. These findings suggest that mutant SOD1 has prion-like attributes that do not extend to SOD1 in sALS tissues.

The Phylogeny and Active Site Design of Eukaryotic Copper-only Superoxide Dismutases.

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or "pseudofungi" species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.

KDM2B Recruitment of the Polycomb Group Complex, PRC1.1, Requires Cooperation between PCGF1 and BCORL1.

KDM2B recruits H2A-ubiquitinating activity of a non-canonical Polycomb Repression Complex 1 (PRC1.1) to CpG islands, facilitating gene repression. We investigated the molecular basis of recruitment using in vitro assembly assays to identify minimal components, subcomplexes, and domains required for recruitment. A minimal four-component PRC1.1 complex can be assembled by combining two separately isolated subcomplexes: the DNA-binding KDM2B/SKP1 heterodimer and the heterodimer of BCORL1 and PCGF1, a core component of PRC1.1. The crystal structure of the KDM2B/SKP1/BCORL1/PCGF1 complex illustrates the crucial role played by the PCGF1/BCORL1 heterodimer. The BCORL1 PUFD domain positions residues preceding the RAWUL domain of PCGF1 to create an extended interface for interaction with KDM2B, which is unique to the PCGF1-containing PRC1.1 complex. The structure also suggests how KDM2B might simultaneously function in PRC1.1 and an SCF ubiquitin ligase complex and the possible molecular consequences of BCOR PUFD internal tandem duplications found in pediatric kidney and brain tumors.